Computational protocol: Virus Specific Differences in Rates of Disease during the 2010 Dengue Epidemic in Puerto Rico

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Protocol publication

[…] Serum specimens with DENV-1 (n = 7), DENV-2 (n = 2) or DENV-4 (n = 4) detected by RT-PCR were randomly selected from municipalities with the highest incidence of the respective DENV-type and inoculated into cultured C6/36 cells; the presence of virus was confirmed by RT-PCR and indirect immunofluorescence . Isolates were further propagated and viral RNA was extracted from culture supernatants using the M48 BioRobot System (Qiagen; Valencia, CA). The envelope glycoprotein (E) gene was amplified and sequenced; sequence data were restricted to the E gene open reading frame (1,485 basepairs). Multiple sequence alignment was performed using MUSCLE available in MEGA 5 (megasoftware.net) and GTR+Γ+I4 was selected as the best nucleotide substitution model as determined by MODELTEST v3.7. Genetic relatedness was inferred and represented with phylogenetic trees using the maximum likelihood method in MEGA 5. MCMC was run in BEAST v1.6.1 (beast.bio.ed.ac.uk) under Bayesian skyline prior, constructed in TreeAnnotator found in the same BEAST package, and visualized in FigTree v1.3. Both trees rendered almost identical tree topologies, therefore confirming genetic relatedness. Evolutionary distances were corroborated by pairwise alignment in BioEdit v7.1.3 and E gene sequences from GenBank were included in the phylogenetic tree to support tree topology by currently circulating genotype. Tree topology was supported by bootstrapping with 1,000 replicates. Genotypes were referred to by previously described nomenclature , . […]

Pipeline specifications

Software tools ModelTest-NG, BEAST, FigTree, BioEdit
Application Phylogenetics
Organisms Homo sapiens
Diseases Death, Dengue, Fever, Infection